Monitoring enzymatic process

ABSTRACT

Techniques, apparatus and systems are described for performing label-free monitoring of processes. In one aspect, a label-free monitoring system includes an array of label-free optical sensors to detect an optical signal in response to synthesis of one or more target genetic structures. Each label-free optical sensor is functionalized with a respective target genetic structure. The system also includes a fluid flow control module that includes fluid receiving units to provide paths for different fluids to flow into the fluid flow control module and at least one switch connected to the fluid receiving units to selectively switch among the fluid receiving units to receive a select sequence of the fluids through the fluid receiving units. The select sequence of the fluids includes at least a dNTP or base. A fluid channel is connected between the fluid flow control module and the array of sensors to allow the select sequence of the fluids to flow from the fluid flow control module to the array of label-free optical sensors.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/746,747, filed Nov. 8, 2010, and entitled “LABEL-FREE OPTICALSENSORS,” which is a 371 National Phase of International PatentApplication PCT/US2008/085988, filed Dec. 8, 2008, and entitled“MONITORING ENZYMATIC PROCESS,” which claims priority to U.S.Provisional Patent Application 61/005,372, filed Dec. 6, 2007, andentitled “METHOD AND APPARATUS FOR CLOCKED SYNTHESIS OF GENETIC MATTER.”The foregoing applications and any and all applications for which aforeign or domestic priority claim is identified in the Application DataSheet as filed with the present application are hereby incorporated byreference in their entireties under 37 CFR 1.57.

BACKGROUND Field

This document relates to label-free sensing of chemical and biologicalmaterials and applications of such label-free sensing.

Description of the Related Art

Various sequencing techniques use a label to attach to a molecule andthe labeled molecule is monitored and interrogated to identify whichbase has been added or removed from a strand of nucleic acid (NA). Suchlabeling can be achieved by various labeling techniques, includingmolecular labeling based on radioactivity, fluorescence, andchemiluminescence. However, a label may cause undesired effects, such asaltering the molecular binding kinetics, interfering with the accuracyof the reaction, and limiting the length of a contiguous readout, andmay require multiple readouts to construct a high confidence sequence.In addition, molecular labeling may require numerous processing stepssuch as label attachment, washing, label removal, scanning, etc. andthus could complicate the process, require extended time for processingand add significant cost.

SUMMARY

Techniques, apparatus and system are described to provide label freesensors used to monitor enzymatic processes. Such label free sensors canbe used to detect sequencing of nucleic acid, for example.

In one aspect, a label-free enzymatic process monitoring system includesan array of label-free optical sensors to detect an optical signal inresponse to modification of one or more target genetic structures byaddition of a base by synthesis. Each label-free optical sensor isfunctionalized with a respective target genetic structure. The systemincludes a fluid flow control module that includes fluid receiving unitsto provide paths for different fluids to flow into the fluid flowcontrol module. The fluid flow control module includes at least oneswitch connected to the fluid receiving units to selectively switchamong the fluid receiving units to receive a select sequence of thefluids through the fluid receiving units. The select sequence the fluidsincludes at least a nucleotide base or deoxyribonucleoside5′-triphosphate (dNTP). A fluid channel is connected between the fluidflow control module and the array of label-free sensors to allow theselect sequence of the fluids to flow from the fluid flow control moduleto the array of label-free optical sensors.

Implementations can optionally include one or more of the followingfeatures. The array of label-free optical sensors can include an opticalevanescent field sensor to hold the respective target genetic structurewithin an evanescent field. The label-free optical evanescent fieldsensor can include a resonant cavity. The resonant cavity can include aring resonator cavity. The array of label-free optical sensors canmeasure a shift in a resonant frequency of the resonant cavity. Thearray of label-free optical sensors can measure a change in a complexrefractive index of the resonant cavity. The fluid flow control modulecan provide a single species of dNTP or nucleotide to the array oflabel-free optical sensors. The fluid flow control module can provide areagent for modifying the target genetic structure to the array ofsensors. The array of label-free optical sensors can detect the opticalsignal while adding the nucleotide base.

In another aspect, sequencing nucleic acids includes functionalizing asurface of a label-free optical sensor with unknown species of nucleicacid. A reagent comprising synthesis materials and a known nucleotidebase is selectively introduced to the unknown species of nucleic acid. Achange in an output signal of the label-free optical sensor is measuredto detect synthesis of the nucleic acid when a nucleotide base in theunknown species of nucleic acid reacts with the known dNTP or nucleotidebase. A next nucleotide base in the unknown nucleic acid to react isidentified based on the introduced known dNTP or nucleotide base and themeasured change in the output signal.

Implementations can include one or more of the following features. Amagnitude of the output signal can be measured to determine a number ofthe introduced known nucleotide base incorporated during the detectedsynthesis. The label-free optical sensor that includes an opticalresonator can be used to monitor the synthesis process occurring withinan optical field of the resonator. The unknown species of nucleic acidcan be amplified using a selectively bound primer and hybridizationsequences. Solid phase amplification and hybridization of the unknownspecies of nucleic acid can be performed in parallel. An amount of theunknown species of nucleic acid can be measured based on the outputsignal of the optical sensor before and after functionalization. A knownsequence of nucleotide bases can be applied and inadvertently ornon-selectively bound materials can be removed by applying a washingagent between the nucleotide bases. The surface of the optical sensorcan be functionalized with a single species of nucleic acid based on theoutput signal of the optical sensor. Measuring a change in an outputsignal of the label-free optical sensor can include: measuring an outputsignal of the label-free optical sensor before introducing the knownnucleotide base; measuring another output signal of the label-freeoptical sensor after introducing the known nucleotide base; andidentifying a difference between the measured output signals. Theunknown species of nucleic acid can be held within an evanescent field.

Yet in another aspect, monitoring an enzymatic process within an opticalfield of a label-free optical resonator includes detecting an opticalsignal from the label-free optical resonator in response to anapplication of one or more enzymes to identify an enzymatic process thatresults in a modification of the nucleic acid. The enzymatic process caninclude one of the following reactions: polymerase driven baseextension; polymerase repair activity driven base excision; reversetranscriptase driven DNA extension; reverse transcriptase driven RNAexonuclease activity; DNA cleavage driven by site specific endonucleaseactivity; annealing driven by ligase enzyme activity and ortopoisomerase action and or recombination enzymes; phosphorylationdriven by kinase; dephosphorylation driven by phosphatase; RNA Splicingdriven by splicing enzymes and or catalytic RNA splicing fragments; andcleavage through miRNA driven DICER complex.

Yet in another aspect, a label-free enzymatic process monitoring systemcan include an array of label-free optical sensors to detect an opticalsignal in response to modification of one or more target geneticstructures. Each label-free optical sensor holds a respective targetgenetic structure within an evanescent field. The system includes afluid flow control module to receive one or more fluids comprising areagent to modify the one or more target genetic structure. A fluidchannel is connected between the fluid flow control module and the arrayof label-free sensors to allow the one or more fluids to flow from thefluid flow control module to the array of label-free optical sensors.

The techniques, apparatus and system as described in this specificationcan potentially provide one or more of the following advantages. Forexample, the amount of target material on a sensor can be measured toallow accurate calibration of the amount on the sensor. Also, the systemcan evaluate when the addition of a base has run to completion. To speedthe synthesis reaction rate, real time monitoring of the reaction can beperformed. In addition, the real time incorporation of bases in asequence extension reaction can be performed based on small sensor sizeand high sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a cross-section of an exemplary optical evanescentfield sensor suitable for sequencing applications.

FIG. 2 illustrates a perspective cross section of another example of anoptical sensor having a ring resonator cavity and a coupling waveguide,formed on a silicon substrate.

FIG. 3a illustrates a top down view of another example of an opticalsensor that includes a ring resonator cavity and two coupling waveguidesin evanescent coupling to the ring resonator cavity.

FIGS. 3b-3d illustrate examples of non-circular shaped ring resonantcavities.

FIG. 4a illustrates a schematic of an example of a synthesis system witha fluid flow control module and a sensor array based on label-freesensors.

FIG. 4b illustrates another example of a synthesis system.

FIGS. 5a-d illustrate several options for sequence of the differentsolutions which can be applied over the sensors.

FIG. 6 shows an example process for synthesizing a nucleic acid.

FIG. 7 shows an example process for monitoring an enzymatic processwithin an optical field of a label-free optical resonator.

These fluid options and sequences are for illustrative purposes, and canbe combined in ways that employ one or more of these approaches in avariety of different orders and combinations.

DETAILED DESCRIPTION

Label-free techniques can provide molecular sensing and detectionwithout using a molecular label. Such label-free techniques can be usedto mitigate certain undesired effects in molecular labeling. Forexample, time consuming and potentially side-effect causinglabel-related process steps can be eliminated. In particular, label-freetechniques can be used to run a synthesis reaction at a substantiallyhigher rate than that of a synthesis reaction based on molecularlabeling. As such, the processing time of a label-free technique may bereduced to a time determined by the kinetics of the synthesis reaction.For example, a label-free technique may be used to reduce the time from10s of minutes per read in a system based on molecular labeling down toseconds, or even milliseconds per base call.

Techniques, systems and apparatus as described in this specification canbe used to provide label-free sensors for monitoring enzymaticprocesses, such as synthesis of genetic material. A resonant cavity withan evanescent field can be used to sequence an unknown nucleic acidsequence without labels. In one aspect, genetic material is held withinthe evanescent field of the resonant cavity and chemical precursors forthe extension of nucleic acid base pairs are added repetitively insequence. The sensor is interrogated synchronously with the addition ofeach subsequent nucleic acid based. A change in the resonant cavityproperties that corresponds to the addition of a particular baseindicates incorporation into the synthesis product and indicates thenext corresponding base.

Examples of label-free techniques, systems and apparatus are describedbelow for sequencing a nucleic acid. For example, a label-freesequencing apparatus can include one or more label-free sensors forsensing a biological and chemical material, a mechanism for holding anucleic acid in interaction with a label-free sensor, a means forcontrollably introducing a reagent and components for modification ofthe nucleic acid, and a label-free means for interrogating the one ormore label-free sensors to obtain output from the one or more label-freesensors and evaluating whether a nucleic acid in interaction with asensor is modified. Such a label-free sensor may be implemented toachieve a limit of detection at or below the addition of a single base.

As a specific example, such a label-free sensor can be implemented byusing an optical sensor that monitors the physical presence of a basevia detection of the optical evanescent wave to determine synthesis, anddoes not require a label attached to the base. In the above exemplaryapparatus, a nucleic acid is placed in the optical evanescent field of alabel-free optical sensor. In another example, a method for sequencingnucleic acids can be implemented based on one or more label-free opticalsensors. In this method, a nucleic acid species is placed within therange of an optical evanescent field sensor and a reagent containingsynthesis materials and a known dNTP or base are introduced to the boundspecies. The output from the optical evanescent field sensor ismonitored to measure a change and the measured change is used todetermine whether synthesis has occurred. This method also includesdetermining the next base in sequence based on knowledge of which dNTPor base is present at the time a sensor signal from the opticalevanescent field sensor indicates the presence of an additional boundmatter.

In one implementation of a label-free sequencing apparatus, an opticalsensor is placed on a substrate in such a manner that the optical sensorcan be interrogated while simultaneously allowing a reaction to occur inthe sensing region of the optical sensor. The optical sensor can beimplemented using an evanescent field sensor. Examples of an evanescentfield sensor include: resonant cavities, Mach-Zehnder interferometers,or other applicable interferometers with a sensing mechanism thatinvolves a change in the complex refractive index in the optical path.One example is a ring resonator, which can be addressed using waveguidesthat are routed out of the sensing region.

An optical ring resonant cavity forms a closed-loop waveguide. In theoptical ring resonant cavity, light propagates in the form of whisperinggallery modes (WGMs) that result from the total internal reflection ofthe light along the curved surface of the ring. The WGM is a surfacemode that circulates along the ring resonator surface and interactsrepeatedly with any material (e.g., target genetic material) on thesurface through the WGM evanescent field. Unlike a straight waveguidesensor, the effective light-material interaction length of a ringresonator sensor is no longer determined by the sensor's physical size,but rather by the number of revolutions of the light supported by theresonator, which is characterized by the resonator quality factor, orthe Q-factor. The effective length L_(eff) is related to the Q-factor byequation 1 below.

$\begin{matrix}{L_{eff} = \frac{Q\; \lambda}{2\pi \; n}} & (1)\end{matrix}$

Where λ is wavelength and n is the refractive index of the ringresonator. Due to the large Q-factor, the ring resonant cavity canprovide sensing performance superior to a straight waveguide sensorwhile using orders of magnitude less surface area and sample volume. Inaddition, the small size of the ring resonator allows an implementationof a larger number of ring resonant cavities in an array of sensors.

An optical sensor on a substrate can be fabricated using a lithographictechnique. Bounding the optical sensor to a substrate can provide aconvenient means to handle the optical sensor and to fabricate multiplesensors in arrays. In other designs, an optical sensor may be detachedfrom a substrate and be free floating.

FIG. 1 illustrates a cross-section of an exemplary optical evanescentfield sensor suitable for sequencing applications. This sensor includesan optical resonator or an optical interferometric structure thatincludes a waveguide 102 formed on a substrate 106 which may be, forexample, a silicon substrate. A first, lower cladding layer 101 with anindex less than that of the waveguide 102 is formed on the substrate 106and is located beneath the waveguide 102. A second, upper cladding layer103 is formed over the waveguide 102 and has an index less than that ofthe waveguide 102. The upper cladding layer 103 is patterned to have oneor more regions 103A in which the cladding material for the uppercladding layer 103 is removed to form a sensing region 103A. The sensingregion is structured to either completely expose a section of thewaveguide 102 or to have a thin layer of the cladding material, to allowa sufficient amount of the optical evanescent field of the guided lightin the waveguide 102 to be present in the sensing region 103A. A geneticmaterial 104 (e.g., DNA, RNA, LNA, etc.) is deposited on a surface via afunctionalizing process in the sensing region 103A in proximity to thewaveguide 102, in such a manner that the evanescent field of thewaveguide 102 can interact with the genetic material 104. Claddingregions in the upper cladding layer 103 are shown to define oneexemplary sensing region 103A that determines which portion of thewaveguide 102 is to be functionalized with the genetic material 104. Aflow channel or fluidic cavity 105 is formed on top of the sensor and afluidic control mechanism is provided to direct different solutions intothe flow channel or fluidic cavity 105 during a sequencing process forsynthesizing a target genetic structure, such as a single speciesnucleic acid in a sensing region 103A. In addition, the fluidic controlmechanism can direct the solutions into the flow channel or fluidiccavity 105 for other enzymatic processes.

FIG. 2 illustrates a perspective cross section of another example of anoptical evanescent field sensor having a ring resonator cavity 203 and acoupling waveguide 202, formed on a silicon substrate 106. Thewaveguides 202 and 203 are displaced from the substrate via a buriedinsulator layer 101 as the lower cladding layer, which may be, forexample, silicon dioxide. Functionalization can occur in proximity tothe surface(s) of the ring resonator cavity 203. In one implementation,similar to the design in FIG. 1, an upper cladding layer over the ringresonator cavity 203 can be patterned to form sensing regions inproximity to the surface of the ring resonator cavity 203 for thesynthesis of a target genetic structure, such as a substantially singlespecies nucleic acid.

FIG. 3a illustrates a top down view of another example of an opticalevanescent field sensor that includes a ring resonator cavity 203 andtwo coupling waveguides 301 and 302 in evanescent coupling to the ringresonator cavity 203. An upper cladding layer 103 is formed over thefirst waveguide 301 and is patterned to define one or more sensingregions above the first waveguide 301 as shown in FIG. 1. The claddinglayer 103 can be used to confine the interaction of the genetic materialin each sensing region to be solely to the immediate proximity of thering 203. The second waveguide 302 is an optical waveguide and may beused to guiding light in connection with the evanescent sensing at asensing region in the first waveguide 301.

The ring resonant cavity 203 of FIGS. 2 and 3 can be formed by awaveguide in a closed loop in various configurations. In FIG. 3a , thering resonator cavity is a closed waveguide loop of a circular shape.This circular closed waveguide loop can support one or more whisperinggallery modes along the circular path of the closed waveguide loop atand around the outer surface of the circular waveguide and may beindependent of the inner surface of the circular waveguide because thewhispering gallery mode exists at and around the outer surface of thecircular waveguide. The optical input to the ring resonant cavity 203can be achieved via evanescent coupling between the waveguide 301 andthe ring resonant cavity 203 which are spaced from each other. In otherimplementations, the closed waveguide loop may be in a non-circularshape that does not support a whispering gallery mode. FIGS. 3b, 3c and3d show example shapes of non-circular ring resonant cavities whichoperate based on the waveguide modes rather than whispering gallerymodes. A waveguide mode is supported by the waveguide structureincluding both the outer and inner surfaces as the waveguide boundariesand thus is different from a whispering gallery mode. Each ring resonantcavity is spaced from the waveguide 201 by a distance d that is selectedto provide desired evanescent coupling. The evanescent couplingconfiguration is indicated by the numeral 320. One aspect of such anon-circular closed waveguide loop forming the ring resonant cavity isto provide the same evanescent coupling configuration 320 whileproviding different closed loop waveguides. FIG. 3b and FIG. 3c show aring resonant cavity in an elliptical shape in a waveguide mode in twodifferent orientations 310 and 320. The specific geometries of theclosed waveguide loop can be selected based on the need of a specificsensor design. Race-track shaped closed waveguide loop, for example, maybe used. FIG. 3d shows an example where the closed waveguide loop 340has an irregular shape that can be designed to fit on a chip. A ringresonant cavity may be used to achieve a high Q factor in the ringresonant cavity in part due to re-circulation of the guided opticalsignal and such a high Q factor can be exploited to achieve a highdetection sensitivity in detecting a minute amount of a material on thesurface of the ring resonant cavity in a label-free enzymatic processbased on optical sensing and monitoring.

FIG. 4a illustrates a schematic of a monitoring system with a fluid flowcontrol module 420 and a sensor array 409 based on label-free sensors.The fluid flow control module 420 includes fluid receiving units, suchas ports 402, 403, 404, 405, 406 and 407 to receive various fluid typesinto the fluid flow control module. Also, one or more switches 401 areprovided in the fluid flow control module to selectively switch-in orreceive one or more of the fluid types into the fluid flow controlmodule. The sensor array 409 includes a matrix of label-free sensors 411arranged in various configurations. For example, the label-free sensors411 can be arranged in a square or rectangular configuration with Nnumber of rows and M number of columns of sensors. The label-freesensors 411 can be arranged in other configurations, such as a circle ora triangle. The label-free sensors 411 may be optical sensors based onthe sensor examples in FIGS. 1-3 b and other sensor designs.

The fluid flow control module 420 is connected to the sensor array 409using a flow channel 408. Solutions in the fluid flow control module 420can flow through the flow channel 408 and arrive at the sensor array409. Different solutions can be obtained in the fluid flow controlmodule 420 by receiving the various fluid types by using the switch 401,and mixing the received fluids. For example, a mix of the variousnucleic acids and the associated synthesis compounds can be addedthrough ports 402-405. In addition, various washing and cleaningsolutions, such as buffers can be switched in through ports 406 and 407.The amount and type of fluids to receive and mix in the fluid flowcontrol module 420 can be controlled using the one or more of theswitches 401. After the fluids are combined and mixed in a junctionregion in the fluid flow control module 420, the resultant solution canbe applied through the fluid channel 408 and over the sensor array 409.In this configuration, each label-free sensor could have a differentunknown sequence attached.

The solution from the fluid flow control module 420 flows over thesensor array 409 and exits the system through the fluid exit 410. Thus,a continuous flow of solutions can be provided across the sensor array409. In some implementations, the solution can be held static in thesensor array 409 by stopping the flow.

FIG. 4b shows another example of a monitoring system with a fluid flowcontrol module 420 and a sensor array 409 based on label-free sensors.Each of the fluid input units 402, 403, 404, 405, 406 and 407 isconnected to a respective switch 401. To selectively input a fluid typethrough one of the fluid input units 402, 403, 404, 405, 406 and 407,the respective switch is used. Remaining components of the monitoringsystem are similar to the system shown in FIG. 4 a.

In the label-free sensors of the sensor array 409, the sensor surfacecan be functionalized to have a target genetic structure, such as anucleic acid sequence held within an optical mode, for example byattachment to the sensor surface. Functionalizing the sensor surface canbe accomplished by various surface chemistry techniques. A single strandor a small number of strands can be attached to the sensor surface. Oncethe sensor surface is functionalized with a strand or strands of thetarget nuclei acid sequence, solid phase synthesis can be performed.

In some implementations, the monitoring system of FIGS. 4a and 4b can beused to functionalize the sensor surface by attaching the species of thetarget nucleic acid sequence in large numbers. To achieve large numbersof the species, the desired species can be purified and amplified, asneeded. The NA can be covalently linked, hybridized to a template, orheld by binding to a protein. The NA can further be held directly on thesurface, or held in a porous film, such as a gel, hydrogel or sol-gel.

For solid phase synthesis, the monitoring system of FIGS. 4a-b can beused to amplify the target sequence only in the active sensing region ofthe sensor. To amplify the target sequence only, selectively boundprimer and hybridization sequences can be used. This can be achievedusing synthesis in situ, photopatterning, or masking techniques forexample. Photopatterning of the primer could be achieved usingultraviolet (UV) sensitive binding chemistry. The desired selectivitycould be achieved by using a mask layer formed out of a material thatdoes not allow surface binding, such as a Teflon based material. Thismaterial itself can be patterned using lithographic approaches and othertechniques.

The monitoring systems of FIGS. 4a-b can be used in applications wherethe target sequence is bound in sufficiently large numbers, and anactive surface is provided for binding only in the region of the sensor.This surface treatment can be more generic and may not need to containprimers or specific hybridization sequences. However, for someapplications, providing a hybridization sequence for a known portion ofa target molecule may be advantageous.

For example, in the case of a single nucleotide polymorphism assay, mostof the target sequence is known, and a hybridization probe could bedesigned to pull down the particular piece of nucleic acid of interest.Then, sequencing can take place on the unknown region to exposeadditions, deletions, substitutions, and other mutations of interest.

The monitoring system of FIGS. 4a-b can be used to provide a combinationof multi-strand attachment and an amplification process for themulti-strand attachment. For example, hybridization of a sample could beobtained using known probe sequences, and then solid phase amplificationcould be performed to boost the numbers.

In some implementations, the solid phase amplification could beperformed in solution, perhaps in real time, while hybridization isoccurring on the sensors. For example, the sensors in the synthesissystem can be placed in a solution undergoing standard Polymerase ChainReaction (PCR) that performs amplification.

By using these techniques for target NA attachment to the sensor, theamount of target material can be measured. This allows accuratecalibration of the target material amount on the sensors. Such accuratecalibration of the target material amount allows normalization of thesubsequent synthesis reactions, and allows a user to determine when anappropriate quantity of target sequence has been accumulated to proceedwith synthesis. This can be observed in real time by making a rapidsuccession of measurements during the target NA attachment and/oramplification process. Also, measurements can be made at significantpoints in the process, such as between PCR cycles. With knowledge of thesensor response prior to attachment and during/after attachment, theamount of target material attached can be determined.

In the monitoring systems of FIGS. 4a-b , any inadvertently ornon-selectively bound materials can be removed from the sensor surface.The removal can be achieved by any of a number of well-known techniques,such as washing and modification of the astringency of the sensor, forexample but not limited to heating and/or changing the saltconcentration or pH of the ambient solution.

Real-time examination of the sensors can provide information regardingthe progress of the removal process. In addition, feedback informationcan be provided to determine when to stop washing, or when to stopheating the target material. For example, the ambient solution can beheated until the non-selectively bound material has been melted off thesensors. However, the temperature is kept below a point where the targetsequence would be completely removed. By watching the rate at which thenon-desired material is removed, an assessment of which temperature touse, and when to stop heating could be accomplished. A similar approachcould be used to regulate the number of wash cycles.

The monitoring systems of FIGS. 4a-b can be used in applications where alarge number of different target species are present in the sameanalyte. The system can be used to control the surface attachmentconditions to deposit only one species on each sensor. For example, thesensor array 409 can be designed to include multiple sensors with eachsensor masked in such a manner to provide surface attachment on thesensor. The concentration, reaction time, temperature, etc., can bemodified to statistically allow only a single target molecule to depositwithin each sensor region. Subsequent solid phase amplification canincrease the number of target molecules up to an appropriate level forobservation of synthesis.

The potential issues with this single species attachment approachinclude the possibilities that more than one species is deposited on asingle sensor, or no deposition occurs on a particular sensor. Both ofthe non-single species cases can be screened for during synthesis. Forexample, when more than one species attach to a single sensor, anirregular sensor response is obtained because only a fractionalproportion of sites is available for the addition of a particular base.This reduction of available sites can result in a fractional sensorresponse in comparison to a uniformly hybridized sensor. Sensors with notarget molecules provides little to no response. In such manners, a goodsensor (single species attachment) can be distinguished from a badsensor (multiple of no species attachment) during the course of thesynthesis process.

After target molecule attachment, the remainder of active surfacebinding sites can be removed or blocked to prevent the accumulation ofnon-selectively bound material. Because the non-selectively boundmaterial can inhibit the accuracy of measurement, blocking the remainingactive surface binding sites can provide a more accurate result.

The monitoring systems of FIGS. 4a-b can be used to perform NA synthesisusing a number of techniques. For example, a polymerase and itsassociated buffer solutions can be used to perform the synthesis. Whenthe sensors of the synthesis system include an evanescent field sensor,the polymerase and buffer solutions can cause a measurable offset to thesensor response. This offset can be addressed in a number of ways. Thesynthesis can be performed in conditions that encourage a steady statepolymerase attachment condition. The steady state polymerase attachmentresults in a steady offset, and the desired synthesis signal is thedelta off of this baseline offset. The baseline offset may change as thesynthesis processes.

Also, the polymerase can be driven off of the target sequence to performthe read, and then reattached when proceeding to subsequent baseaddition. When the polymerase is detached, the polymerase can remain inthe ambient solution or washed away from the sensor region and thensubsequently replaced. For a single synthesis step, such as that neededin a single nucleotide polymorphism (SNP) reaction, removing thepolymerase is not critical because subsequent reattachment is notnecessarily required. When large numbers of bases are to be synthesized,then it is advantageous to keep the polymerase attached, if possible.

The monitoring systems of FIGS. 4a-b can be used to detect a geneticvariation called Single Nucleotide Polymorphisms (SNPs). SNPs arecommonly determined through use of hybridization arrays containing eachof the 4 possible variants as part of a 25-mer strand of DNA. To detectSNPs, appropriately prepared DNA is exposed to the hybridization arrays.An array element with the strongest binding indicates the SNP typepresent. In the monitoring systems of FIGS. 4a-b , a hybridization arrayis prepared on a label-free sensor, where the hybridization sequence isdesigned to bind to the DNA proximal to the SNP location, but leavingthe SNP base exposed for sequencing by synthesis. The hybridizationarray can be designed on the label-free sensor such that the SNP is thenext base in sequence. Also, the hybridization array can be designed onthe label-free sensor such that the SNP is a known number of bases fromthe termination of the hybridization sequence. In either case,sequencing by synthesis is performed as described above, and theidentity of the SNP can be determined based on sensor response.

The monitoring systems of FIGS. 4a-b can be implemented to allowdifferent bases to flow sequentially over the sensors. The monitoringsystems of FIGS. 4a-b can be designed to pump different bases insequence over the sensors. The flow can be continuous, or it can bestopped once the desired mixture is over the sensor region. Between thedifferent solutions containing the different bases, a number of otherfunctional solutions can be added.

FIGS. 5a-d illustrate examples of a sequence of different solutionswhich can be applied over the sensors in a sensor array. FIG. 5a shows abasic configuration of a synthesis mixture that allows addition of dNTPsor NA bases in sequential order. In this example, the dATP or base A isapplied over the sensors (e.g., sensors in the sensor array 409 of themonitoring systems) and a measurement is made. After the bases or dATP,dCTP, dTTP, dGTP, etc. are sequentially applied over the sensors. Thesynthesis enzyme (such as polymerase) may be optionally added with eachdNTP or base. The synthesis enzyme can be added prior to the dNTP orbase if the synthesis enzyme maintains a steady or otherwise predictablebehavior. Also, the synthesis enzyme can be included with all solutionsto guarantee the presence of the enzyme.

The sequence of solution in FIG. 5b shows a buffer solution, B, appliedbetween the different dNTPs or bases. For example, the sensors can bewashed between dNTPs or bases. A washing solution, such as a buffer, B,can be added to the sequence of reagents applied over the sensors,potentially between each successive dNTP or base nucleotide solution.The buffer solution, B, is applied to ensure that the previous dNTP orbase has been swept or washed from the reaction region prior to additionof the next dNTP or base. For example, the buffer solution is appliedbetween application of dATP or base A and buffer B to prevent or reducecommingling of dATP and B (or bases A and B). This ensures that eachbase addition is separated by time and solution and thus can beisolated. The buffer solution can also be used to remove non-selectivebinding.

In addition, non-mixing regions can be added in the synthesis system toprevent different base solutions from intermixing. This could beaccomplished by applying a bubble of air or other non-mixing fluidinjected in series with the reagents. Also, a sufficiently large amountof the new type of dNTP or base solution can be applied to guaranteeremoval of the previous dNTP or base solution.

Even small amounts of the previous base solution remaining in the sensorcan become an issue, despite the fact that the previous synthesis stepshould have been fully reacted. If the new dNTP or base reacts, it ispossible that the next subsequent dNTP or base will be next in line toreact, and thus, a small number of strands will have skipped ahead onebase. This anomaly can be minimized by thorough removal of the old basesolution (e.g., by using the washing solution) prior to introduction ofthe next one.

The sequence of solutions in FIG. 5c shows adding and removing asynthesis enzyme prior to each base inquiry. For example, a synthesisenzyme, such as polymerase, P, can be added prior to each dNTP or base.The added polymerase, P, is removed before the next dNTP or base isadded using a solution that encourages the disassociation of thepolymerase with the target strand, RP. Also, after removing thepolymerase, another polymerase is added before the next dNTP or base.For example, FIG. 5 c shows the addition of polymerase, P, before theaddition of dATP or base A. Then, after adding the dATP or base A andbefore adding the next base, dCTP, the added polymerase, P, is removedusing RP. Then another polymerase, P, is added before the dCTP or base.This adding and removing of the polymerase is repeated before additionof each dNTP or base.

The sequence of fluids in FIG. 5d shows a polymerase and associated dNTPor base that are added to obtain a synthesis reaction (A+P). The addedpolymerase is removed using a solution that disassociation of thepolymerase with the target strand, RP. Also, a known buffer thatfacilitates more accurate measurement, M, of the sensor is added. Theaddition and removal of the polymerase and the addition of the bufferfor measurement are repeated before addition of each dNTP or base.

In some implementations, these fluid sequence options of FIGS. 5a-d arecombined in ways that employ one or more of these approaches in avariety of different orders and combinations.

Also, a particular dNTP or base can bind a number of times in a rowduring a particular sequence. For example, the sequence of A, A-A orA-A-A can react. Such repeated incorporation by a single dNTP or basecan be determined by measuring the amplitude of the sensor response. Ifthe complete incorporation of a single base results in a certainresponse, the complete incorporation of two bases will approximatelydouble the response and three bases will approximately triple theresponse, etc. Thus by knowing the magnitude of the responsequantitatively, multiple additions of a single base type can bedetermined.

Also, bases that terminate the reaction after the addition of a singlenucleotide can be used. However, the use of terminating bases needsadditional chemistry to process a series of bases in order to allow thereaction to proceed.

In addition, the monitoring systems of FIGS. 4a-b can be used toevaluate when the addition of a dNTP or base has run to completion.Because it is advantageous to speed up the synthesis reaction rate, realtime monitoring of the reaction can be performed. When it is determinedthat the reaction is complete, or that no reaction is going to occur,the process of introducing the next dNTP or base can be started.

As more and more bases are added to the synthesis product, the locationfor the additional base may be moving either closer or further from thesensor surface, depending on what primer configuration is employed andhow the target sequence is attached to the sensors. For an evanescentfield sensor of the monitoring systems shown in FIGS. 4a-b , anon-uniform response can be obtained as a function of proximity to thesensor surface. Thus, the sensor signal can be corrected for thischange. The expected signal baseline response can be adjusted as afunction of time because the response of the signal has been calibratedfor the known location of polymerization reaction or because the signalresponse is being fit to the known field decay.

This approach is applicable for DNA, RNA or any type of nucleic acidcomplex which can by sequentially synthesized. In the case of RNA, aconversion to cDNA might be needed prior to any amplification process,but is not absolutely necessary.

This approach can be scaled to high degrees of parallelism byincorporating a large number of sensors and/or scanning systems. Forexample, many sensors can be employed on a single chip of a synthesissystem. Also, many chips can be implemented in a synthesis system toachieve scaling.

In some implementations, the monitoring systems of FIGS. 4a-b can beimplemented to include a microfluidic switching manifold in immediateproximity to the sensor(s) to allow more rapid fluidic switching times.Such a microfluidic switching manifold can accelerate the sequencingprocess.

In some implementations, monitoring systems of FIGS. 4a-b can be used tomonitor an enzymatic process, other than synthesis described above,within an optical field of an optical resonator. An enzymatic processcan result in a modification of the nucleic acid when one or moreenzymes are applied. Examples of the enzymatic process can include oneor the following reactions: (1) polymerase driven base extension; (2)polymerase repair activity driven base excision; (3) reversetranscriptase driven DNA extension; (4) reverse transcriptase driven RNAexonuclease activity; (5) DNA cleavage driven by site specificendonuclease activity; (6) annealing driven by ligase enzyme activityand or topoisomerase action and or recombination enzymes; (7)phosphorylation driven by kinase; (8) dephosphorylation driven byphosphatase; (9) RNA Splicing driven by splicing enzymes and orcatalytic RNA splicing fragments; and (10) cleavage through miRNA drivenDICER complex.

FIG. 6 shows an example process for synthesizing a nucleic acid.Label-free optical sensors are functionalized with a single species ofan unknown nucleic acid (602). A reagent comprising synthesis materialsand a known dNTP or base is selectively introduced to the unknownspecies of nucleic acid (604). A change in an output signal of thelabel-free optical sensor is measured to detect synthesis of the nucleicacid when a nucleotide base in the unknown species of nucleic acidmatches with the known dNTP base (606). A next nucleotide base in theunknown nucleic acid to react is identified based on the introducedknown dNTP or base and the measured change in the output signal (608).This process can be repeated by applying a sequence of dNTPs or base asshown in FIGS. 5a -d.

Also, a magnitude of the output signal can be measured to determine anumber of the introduced known dNTP or base incorporated during thedetected synthesis. The unknown species of nucleic acid can be amplifiedusing a selectively bound primer and hybridization sequences. Solidphase amplification and hybridization of the unknown species of nucleicacid can be performed in parallel. An amount of the unknown species ofnucleic acid can be measured based on the output signal of the opticalsensor before and after functionalization. Not just one but a sequenceof known dNTPs or bases can be applied and inadvertently ornon-selectively bound dNTPs or bases can be removed by applying awashing agent between the dNTPs or bases. The surface of the opticalsensor can be functionalized with a single species of nucleic acid basedon the output signal of the optical sensor. Further, measuring a changein an output signal of the label-free optical sensor can include:measuring an output signal of the label-free optical sensor beforeintroducing the known dNTP or base; measuring another output signal ofthe label-free optical sensor after introducing the known dNTP or base;and identifying a difference between the measured output signals. Theunknown species of nucleic acid can be held within an evanescent field.

FIG. 7 shows an example process for monitoring an enzymatic processwithin an optical field of a label-free optical resonator. Label-freeoptical sensors can be used to hold a single species nucleic acid withinan evanescent field (702). A reagent for modifying the single speciesnucleic acid is applied to the single species of nucleic acid (704). Anoptical signal from the label-free optical resonator is measured inresponse to the applied reagent to identify an enzymatic process thatresults in a modification of the nucleic acid (706). The enzymaticprocess can include one of the following reactions: polymerase drivenbase extension; polymerase repair activity driven base excision; reversetranscriptase driven DNA extension; reverse transcriptase driven RNAexonuclease activity; DNA cleavage driven by site specific endonucleaseactivity; annealing driven by ligase enzyme activity and ortopoisomerase action and or recombination enzymes; phosphorylationdriven by kinase; dephosphorylation driven by phosphatase; RNA Splicingdriven by splicing enzymes and or catalytic RNA splicing fragments; andcleavage through miRNA driven DICER complex.

In some implementations, one or more evanescent wave sensors includesmultiple nucleic acid bound to the surface of the sensors. The sensorscan include a means for introducing a reagent containing all necessarycomponents for synthesis and a base of choice. The sensors can include ameans for interrogating the sensors and evaluating if matter is bound.

A method for sequencing can include binding multiple known species ofnucleic acid within the range of an evanescent field sensor. A reagentcontaining synthesis materials and a known base are introduced to thebound species. The change in output from the evanescent field sensor isobserved to determine if synthesis has occurred. The next base insequence is determined based on knowledge of which base was present atthe time the sensor signal indicates the presence of additional boundmaterial. The sensor can be a resonant cavity. The resonant cavity canbe a ring resonator. The ring resonator can be made primarily of siliconand the ring is disposed on a silicon-on-insulator wafer.

One or more evanescent wave sensors can include a plurality of asubstantially single species nucleic acid held within the evanescentfield, a means for controllably introducing a reagent and components formodification of the nucleic acid, and a label-free means forinterrogating the sensor and evaluating if the nucleic acid is modified.One or more evanescent wave sensors can include a plurality of asubstantially single species nucleic acid bound to the surface, a meansfor introducing a reagent containing all necessary components forsynthesis, and a base of choice, and a label-free means forinterrogating the sensor and evaluating if matter is bound.

A method for sequencing nucleic acids can include holding a plurality ofan unknown species of nucleic acid within the range of an evanescentfield sensor, introducing a reagent containing synthesis materials and aknown base to the bound species, observing the change in output from theevanescent field sensor to determine if synthesis has occurred, anddetermining the next base in sequence based on knowledge of which basewas present at the time the sensor signal indicates the present ofadditional bound matter.

An optical resonator can be used to monitor an enzymatic processoccurring within the optical field of said resonator. The enzymaticprocess can result in a modification of the nucleic acid, such as:polymerase driven base extension, polymerase repair activity driven baseexcision, reverse transcriptase driven DNA extension, reversetranscriptase driven RNA exonuclease activity, DNA cleavage driven bysite specific endonuclease activity, annealing driven by ligase enzymeactivity and or topoisomerase action and or recombination enzymes,phosphorylation, driven by kinase, dephosphorylation driven byphosphatase, RNA Splicing driven by splicing enzymes and/or catalyticRNA splicing fragments, or cleavage through miRNA driven DICER complex.

While this specification contains many specifics, these should not beconstrued as limitations on the scope of an invention or of what may beclaimed, but rather as descriptions of features specific to particularembodiments of the invention. Certain features that are described inthis specification in the context of separate embodiments can also beimplemented in combination in a single embodiment. Conversely, variousfeatures that are described in the context of a single embodiment canalso be implemented in multiple embodiments separately or in anysuitable subcombination. Moreover, although features may be describedabove as acting in certain combinations and even initially claimed assuch, one or more features from a claimed combination can in some casesbe excised from the combination, and the claimed combination may bedirected to a subcombination or a variation of a subcombination.

Only a few implementations are disclosed. Variations and enhancements ofthe described implementations and other implementations may be madebased what is described and illustrated.

1-22. (canceled)
 23. A method comprising: causing a known sequence ofnucleotides to continuously flow by a label-free optical sensor having asurface functionalized with an unknown species of nucleic acid;measuring changes in an output signal of the optical sensor to detectsynthesis reactions between the unknown species of nucleic acid and theknown sequence of nucleotides; and identifying a sequence of nucleotidesin the unknown species of nucleic acid based on the measured changes inthe output signal and the known sequence of nucleotides.
 24. The methodof claim 23, further comprising discouraging comingling of differentnucleotides in the known sequence of nucleotides.
 25. The method ofclaim 24, wherein discouraging comingling of different nucleotides inthe known sequence of nucleotides comprises providing buffer solutionsin between the different nucleotides.
 26. The method of claim 24,wherein discouraging comingling of different nucleotides in the knownsequence of nucleotides comprises providing air bubbles in between thedifferent nucleotides.
 27. The method of claim 23, further comprisingdetecting repeated incorporation of a nucleotide, from the knownsequence of nucleotides, in the unknown species of nucleic acid based onmagnitude of change in the output signal of the optical sensor.
 28. Themethod of claim 27, further comprising detecting a double incorporationof a nucleotide, from the known sequence of nucleotides, in the unknownspecies of nucleic acid based on a doubled change in the output signalof the optical sensor.
 29. A device comprising: a fluid flow controlmodule configured to cause a known sequence of nucleotides tocontinuously flow by a label-free optical sensor having a surfacefunctionalized with an unknown species of nucleic acid; wherein thedevice is configured to measure changes in an output signal of theoptical sensor to detect synthesis reactions between the unknown speciesof nucleic acid and the known sequence of nucleotides; and wherein thedevice is configured to identify a sequence of nucleotides in theunknown species of nucleic acid based on the measured changes in theoutput signal and the known sequence of nucleotides.
 30. The device ofclaim 29, wherein the fluid flow control module is configured todiscourage comingling of different nucleotides in the known sequence ofnucleotides.
 31. The device of claim 30, wherein the fluid flow controlmodule is configured to provide buffer solutions in between thedifferent nucleotides in the known sequence of nucleotides.
 32. Thedevice of claim 30, wherein the fluid flow control module is configuredto provide air bubbles in between the different nucleotides in the knownsequence of nucleotides.
 33. The device of claim 29, wherein the deviceis configured to detect repeated incorporation of a nucleotide, from theknown sequence of nucleotides, in the unknown species of nucleic acidbased on magnitude of change in the output signal of the optical sensor.34. The device of claim 33, wherein the device is configured to detect adouble incorporation of a nucleotide, from the known sequence ofnucleotides, in the unknown species of nucleic acid based on a doubledchange in the output signal of the optical sensor.